Continuous balanced flow fixed boundary electrophoresis

ABSTRACT

A continuous balanced flow electrophoresis apparatus utilizes a fixed, colloid-permeable boundary membrane across which net liquid flow is minimized. The apparatus is employed in electrophoretic separation of colloids such as blood plasma proteins in which fluid flow rates are controlled and balanced to minimize or prevent liquid transfer across the boundary membrane.

BACKGROUND OF THE INVENTION

The present invention is concerned with separation of suspensions andsolutions by means of electrophoresis. Various types of electrophoreticseparations are known, including continuous free-boundaryelectrophoresis, exemplified by Bier, U.S. Pat. No. 2,878,178;electrodecantation, exemplified by Polson, U.S. Pat. No. 2,801,962; andforced flow electrophoresis, exemplified by Bier, U.S. Pat. No.3,079,318.

Separations utilizing electrophoretic migration of an electricallycharged colloidal component through a colloid permeable membrane areknown and can be carried out on known apparatus after modification inaccordance with the invention. Such apparatus typically comprises acompartment defined by a pair of semipermeable membranes which separatethe compartment from a pair of electrodes, and at least one colloidpermeable boundary membrane which divides the compartment into at leastone pair of cells. Many apparatus will include a stack of cell pairs,each pair being separated by a semipermeable membrane. Appropriate fluidinlets and outlets, and appropriate electrical circuitry are provided topass fluids through the cell pairs and to apply a direct currentelectric field across the permeable membrane. Apparatus of this generaltype is described, for example, by Milan Bier, U.S. Pat. No. 3,079,318.Such apparatus utilizes forced flow electrophoresis, in which thecolloid permeable membrane serves as a filter through which liquid isforced by adjustment of pressure and flow rates through the cells. Suchseparations have been described by Smolka and Logan, PreparativeBiochemistry, 2 (4), 329-45 (1972).

BRIEF SUMMARY OF THE INVENTION

This invention is directed to an improvement in electrophoreticseparation methods and apparatus. More particularly, the invention isdirected to improvements in fixed boundary continuous electrophoreticseparation methods and to apparatus useful for continuous separation ofcolloidal suspensions and solutions. The invention provides apparatuscomprising a compartment defined by semipermeable, non-conductingmembranes, a colloid permeable membrane in the compartment dividing thecompartment into a pair of cells, electrodes for applying a directcurrent electric field across the boundary, a fluid inlet and outletcommunicating with opposite ends of the cell in each pair of cells,means for introducing fluid into each cell, passing fluid along thesurface of the colloid permeable membrane and withdrawing fluid from thecell, and means for balancing the fluid flow on opposite sides of theboundary membrane to minimize or eliminate net fluid transport throughthe colloid permeable boundary membrane.

In the method of the invention a colloidal solution is introduced intoone end of an electrophoresis cell and passed along one face of acolloid permeable non-conducting boundary membrane and withdrawn fromthe other end of the cell at a volumetric rate of aproximately equal toits rate of introduction; a second solution is passed along the oppositesurface of the boundary membrane in a corresponding manner while adirect current electric field is applied across the membrane and the twosolutions, thereby inducing electrophoretic migration of an electricallycharged component of one of the solutions across the boundary, therebyincreasing the concentration of said component in the liquid on theother side of said boundary. At the same time, electrically neutralcomponents and components of opposite charge (to the migratingcomponents) are retained on their original side of the boundary.

The invention can utilize many elements of the cell stack apparatussimilar to those utilized in forced flow electrophoresis orelectrofiltration, however, in the apparatus and process of theinvention net liquid flow through the colloid permeable boundary elementis virtually eliminated. However, many of the known general parametersof electrophoretic separations are applicable to the invention, and canbe dealt with in ways analogous to known methods. For example, there maybe mentioned

1. General relationship of electrophoretic mobility, migration velocityof the charged species and polarity and strength of the electric field;

2. Generation of heat by the electric current and cooling of thecolloidal liquid being treated;

3. Migration of non-colloidal electrolytes through the boundary andbalancing of electrolyte content on opposite sides of the boundary;

4. Spatial configuration of the apparatus for gravitational stability;

5. Generation of gases at the electrodes, and venting of the apparatus;

6. Use of non-conducting materials for the membranes, boundarydiaphragm, enclosure, etc. Such considerations are well known, and arediscussed in the prior art (e.g. Bier U.S. Pat. No. 3,079,318) and neednot be described in detail here.

In balanced flow electrophoresis using the method and apparatus of theinvention it is essential that there be no significant net fluidtransfer across a permeable cell boundary membrane. Thus the inventioncontrasts sharply with forced flow electrophoresis, electrofiltrationand electrodecantation, which require fluid transfer across a boundarymembrane, and with free-boundary electrophoresis, in which the absenceof a membrane inherently allows liquid transfer. The elimination ofliquid transfer across the boundary provides a dual separation andconcentration function. This dual function can be illustrated byconsidering a starting solution, arbitrarily designated A, whichcontains a mixture of components, such as dissolved or colloidalproteins, to be separated. Under the appropriate conditions forelectrophoretic separation, some of the components will migrate acrossthe boundary to a solution on the other side, arbitrarily designated B.Other, differently charged components, e.g., non-migrating species orions migrating away from the boundary, remain in solution A; they can beconsidered non-migrating components with respect to the cell boundary.In a forced-flow electrophoretic separation, some of the non-migratingcomponents are carried across the boundary by fluid transport, e.g. fromsolution A to solution B. This distributes them between solutions A andB, reducing the absolute amount present on both sides of the boundaryand diluting the absolute concentration of migrating components obtainedin solution B. By way of contrast, in the balanced flow electrophoresisof the invention, only the migrating components are moved across theboundary with their resulting concentration and purification in solutionB. Simultaneously, the non-migrating components remain in solution A,being purified by separation from the migrating components. Sinceneither solution is diluted or contaminated by the other, the outputstreams A and B can both be useful products.

A particularly useful embodiment is the separation of gamma globulinfrom albumin in solutions such as blood plasma. It has been found thatexcellent separations can be obtained by subjecting buffered bloodplasma directly to balanced flow electrophoresis. Blood plasma ismodified only by buffering to a pH between the isoelectric points of theproteins to be separated (4.7 for albumin and 7.4 for gamma globulin)and employed as one stream, while a compatible buffer solution isemployed as the other. A gamma globulin output stream and an albuminoutput stream can be obtained simultaneously in a one step continuousprocess, with both products being sufficiently purified to beconcentrated, sterilized or otherwise treated by other continuousprocesses with or without further protein separation steps.

BRIEF DESCRIPTION OF THE FIGURES OF THE DRAWINGS

FIG. 1 is a schematic cross-sectional view of a balanced flowelectrophoresis cell of the invention showing the concurrent directionof flow in the various fluid streams.

FIG. 2 is a schematic cross-sectional view of an electrophoresis cell ofthe invention illustrating a counter flow embodiment.

FIG. 3 is a schematic view of the overall apparatus.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIGS. 1 and 2, the electrophoretic cell stack 4 comprises acell enclosure 5 which can be made of separate sections of anelectrically non-conducting material pressed or clamped together by anysuitable retaining means. Disposed within the enclosure 5 are a pair ofelectrodes, anode 6, and cathode 7. The electrodes 6,7 are mounted inopposite walls of the enclosure 5 and connected by conventional means toa source of direct current for applying the electric field. Also withinenclosure 5 there are disposed a plurality of colloid-impermeablemembranes 8 extending completely across the interior volume of enclosure5 and generally parallel to the electrodes 6,7. Membranes 8 are of asemipermeable, non-conducting material such as cellulose acetatemembranes or the like used in dialysis, permitting small ions such asinorganic salts, phosphate buffers, citrate buffers, etc. to traversethe membrane while preventing colloidal materials such as blood proteinsfrom passing through the membranes 8. The membranes 8 thus define aninner compartment within enclosure 5. A colloid permeable,non-conducting boundary membrane 10 is disposed within enclosure 5between membranes 8, dividing the compartment into a pair of cells.Boundary membrane 10 is of a non-conducting material which is permeableto the substance to be separated or concentrated in the apparatus. Itmay be made of filter paper, filter cloth, ceramic filtering material orthe like.

The enclosure 5 includes an inlet 15 to cell 12, and at the opposite endof cell 12, an outlet 16 for passing a fluid stream, e.g. solution A,through cell 12. In a corresponding manner there is provided an inlet 13to cell 11 and an outlet 14 at the opposite end of cell 11 for passinganother stream, e.g. solution B, through cell 11.

In operation, the electric field may result in the formation ofundesired concentrations of ionic species, and may also result information of gases and heat at the electrodes 6,7. Accordingly, asillustrated in FIG. 2, the electrophoresis stack includes means forintroducing a cooling solution, such as a buffer solution into theregions defined by membranes 8 and enclosure 5, e.g. the regions coolingsolution inlets 18 and outlets 19. To further minimize undesired effectsof the electric field, an additional pair of membranes 9, similar tomembranes 8, can be disposed in the enclosure 5 between each membrane 8and each of the electrodes 6,7, as illustrated in FIG. 1. Appropriateinlets 20, and fluid conduits (illustrated by arrowed lines) and outlets22 are provided for passing a cooling solution between first one pair ofadjacent membranes 8,9 adjacent the anode and then past the cathode 7;and for passing another cooling solution between the pair of adjacentmembrane 8,9 which are adjacent the cathode 7, then past the anode 6.

The cooling solution streams are preferably recycled to theelectrophoretic stack 4, as illustrated in FIG. 3, by passing themthrough cooling means, e.g., a heat exchanger 24, combining the cooledsolutions in a suitable refrigerated vessel, e.g., cooling solutionreservoir 26, and dividing the solution and passing it back to the stack4 by conventional means, e.g. pumps 27. A leveling tank 56 is preferablyincluded in the return line between the stack 4 and reservoir 26.Recombination and mixing of the cooling solution streams outside thecell stack 4 (e.g. in reservoir 26) allows the ionic species formed atthe anode 6 and cathode 7 to neutralize each other chemically.Conventional means for adjusting the pH of the cooling solution can beconveniently provided at reservoir 26.

A preferred form of the overall apparatus is illustrated in FIG. 3. Thecell stack 4 is not illustrated in detail in FIG. 3. The generalfeatures of the cell stack 4 have been described in more detail inreference to FIGS. 1 and 2. If desired, the cell stack 4 can comprise aplurality of cell pairs, each pair having its own permeable boundarymembrane 10, and each pair separated from the adjacent cell pairs andfrom the electrodes by a semipermeable membrane, or preferably by a pairof semipermeable membranes 8,9 to permit a cooling solution to be passedbetween each cell pair in the stack, as well as between the cell pairsand the electrodes.

As illustrated in FIG. 3, a feed reservoir 28 provided with temperaturecontrol means, e.g., cooling coil 29 is provided for a liquid, solutionA, to be separated. A similar reservoir 30 with a cooling coil 31 isprovided for the other fluid, solution B.

The two solutions, A and B, are introduced into the cell stack 5 at acontrolled rate by feed pumps 32, 24; and withdrawn from the stack 5 bya corresponding pair of pumps 36,38. If desired, the processed streams Aand B can be divided downstream of pumps 36,38 so that a predeterminedportion of each stream is returned to its respective reservoir, 28 or30, and the remainder of each stream is collected in a correspondingcollection reservoir 40,42. The proportion of each solution to berecycled is controlled by conventional means, e.g. valves 44 in theappropriate branches of the fluid circuit. When desired, instead ofrecycling the streams A and B, their collection reservoirs can serve asfeed reservoirs for a second cell stack, or other separatory device.

Intermediate the cell stack 5 and pumps 36,38, the apparatus includesmeans for balancing the hydrostatic pressure of solutions A and B, e.g.,leveling tanks 46,48. In a convenient embodiment, the leveling tanks46,48 can be simply a pair of vessels each having an inlet 52 and anoutlet 50. The inlets 52 are connected to the cell stack 5 on oppositesides of the boundary membrane 10. The outlet 50 of each leveling tank46,48 are connected to their respective outlet pumps 36,38. In oneembodiment, the tanks 46,48 are also overflow tanks, i.e., the outlet 50on each tank is vertically higher than its inlet 52. The outlets 50 ofboth tanks are then vertically level with each other to maintain thesame hydrostatic pressure head in both solutions A and B. The pressureof the air in both tanks 46,48 above the liquid level is maintainedequal by conventional means such as venting both tanks to atmosphere.The leveling tank 50 on the cooling solution line operates in the samemanner.

When the leveling tanks 46,48 are arranged as overflow tanks, theapparatus can be operated without the outlet pumps 36,38 if the pumpingpressures provided by the feed pumps 32,34 are kept equal, and the cellgeometry remains constant. When it is desired to pass the two streams Aand B through the device using different pumping pressures or when thecell geometry is subject to change, due to membrane distortion or thelike, the outlet pumps 36,38 must be employed. When the outlet pumps36,38 are in operation, the leveling tanks 46,48 serve only to balancehydrostatis pressure across the boundary membrane 10, and need not serveas overflow tanks. In this mode of operation, the relative height ofinlets 52 and outlets 50 is not critical.

In operation, each of the outlet pumps 36,38 is operated at a volumetricflow rate substantially equal to the flow rate provided by itscorresponding feed pump 32,34 to ensure that the liquid flow rate doesnot affect the hydrostatic pressure of solutions A and B in the cellstack 4. Also, the cell stack 4 itself is oriented so that the boundarymembrane 10 is substantially vertical (as illustrated by the verticallines in FIG. 3) to equalize the hydrostatic pressure across themembrane 10.

OPERATION OF THE LEVELING TANKS AND OUTLET PUMPS

The materials usually employed as semipermeable membranes 8 frequentlyare subject to severe, distortion in use. Even though the membranes 8are fastened to the rigid cell support 5 at their edges, the majorportion of their surface is exposed in the cell without rigid support,and usually wrinkles or buckles severely when wet. These distortionschange the cross-sectional area of the fluid flow path through the cell,resulting is localized erratic variations in liquid pressure as thesolution is pumped through the cell.

The boundary membrane 10 is more porous than membranes 8, and isgenerally of a structurally weaker material such as filter paper orfilter cloth. In use the boundary membrane can bulge or sag to one side,decreasing the flow area and increasing the fluid pressure in one streamwith a corresponding increase in flow area and reduced pressure on theother side. The internal pressure differences resulting from membranedistortions cause undesirable fluid transport across the boundarymembrane, resulting in uncontrolled cross-contamination and dilution bymass transport through the boundary. The membrane distortion effectsbecome more severe as the size of the apparatus is increased, with thecorresponding decreasing ratio of linear edge support to membranesurface area.

In the method and apparatus of the invention, liquid is introduced intoand withdrawn from each cell by a pair of pumps, one upstream and onedownstream of the cell, with both pumps preferably operating at the samevolumetric flow rate. Additionally, a standpipe, or leveling tank 46,48is provided in the flow paths intermediate the pumps, to provideautomatic pressure equalization on both sides of the permeable membrane.The leveling tanks 46,48 are liquid containers each having a liquidinlet and an outlet and having a wall which extends vertically upwardabove the inlet and outlet to permit containment of a fluid columntherein. Each leveling tank 46,48 is thus adapted to function as astandpipe in stabilizing liquid pressure in its respective cell.

In combination with the pumps and with each other, the leveling tanks46,48 automatically balance the pressures on opposite sides of theboundary membrane 10. The apparatus is constructed so that the liquidlevel in each leveling tank is above the uppermost portion of theelectrophoresis cells, and so that the liquid level in one leveling tankcan achieve a pressure-equalizing level relative to the other levelingtank. In most instances, the desired cell geometry and flow rate onopposite sides of the membrane are equal and the liquid levels in theleveling tanks 46,48 will be level with each other under suchconditions.

When a pressure difference develops during operation (due to membranedeformation, change in pumping pressure or the like), there is atransient liquid flow through the boundary membrane 10. The apparatusincludes means for providing uniform flow rate from the leveling tanks,e.g. downstream outlet pumps 36,38. Thus, any such transient liquid flowacross the boundary automatically changes the relative liquid levels inthe leveling tanks 46,48 to re-equalize pressure across the boundarymembrane 10. In continuous electrophoresis, the automatic pressurecompensation provided by the leveling tanks 46,48 and constant flowoutlet pumps 36,38 substantially eliminates net liquid flow across theboundary membrane 10.

In practice, the leveling tanks 46,48 can be relatively small; e.g.tubes about 2 centimeters in diameter and 7 to 8 centimeters in height(above their outlets) have given excellent results with cells in whichthe boundary membrane is about 100 square centimeters in area. Inpractical separation of blood proteins, the flow rate through each cellis generally low, e.g., 0.25 to 2 milliliters per minute, and thepressure differences across the boundary 10 are also low, generally onthe order of 1 to 3 millimeters of water, as indicated by observation ofthe leveling tanks. While the absolute pressure differentials arerelatively slight, the resulting fluid flow can be substantial, due tothe high porosity and area of the boundary membrane 10.

In large scale operations using a cell stack 4 with a large number ofcell pairs, a leveling tank can be economically employed in the outputstream from each cell. By using a conventional peristaltic pump,individual constant flow inlet and outlet pumps can be easily employedfor large numbers of cells. However, it has been found that the requiredbalanced pressure can be achieved by connecting the outlets of aplurality of cells to a single leveling tank and outlet pump. Furtherreduction in pumping capacity has been obtained by providing an outletpump for only one of the two liquid streams, e.g. stream B. In this casethe leveling tank on the other stream, e.g. stream A, can functionadequately as an overflow tank without requiring a second outlet pump.It will thus be apparent that the invention can be adapted to a widevariety of specific embodiments.

It will be apparent that the flow rates of the two solutions, A and B,can be the same or different, as may be desired, without altering thepressure balance in the stack 4 itself. It will also be apparent thatthe equal balancing of liquid pressure across the membrane 10 virtuallyeliminates net fluid transfer across the membrane. Thus mass transportof ions and molecules across the boundary membrane 10 takes place bydiffusion and electrophoretic migration only. Since the electrophoreticfield strength can readily be made the dominant factor, and sincediffusion can be minimized (e.g. by choice of membrane 10, selection ofpH and ionic strength of solutions A and B, etc.) the balanced flowelectrophoresis system is adaptable to easily controllable preciseseparations.

In contrast to forced flow electrophoresis, electrodecantation,electrofiltration and the like, the balanced flow electrophoresisinvention provides a high degree of control over electrically neutralcomponents, i.e., the components which do not migrate under theinfluence of the electric field. The invention can thus be employed toseparate a mixture of components, giving, as one output stream, aproduct with a high concentration of the migrating components of onecharge and, in a second output stream, a second product enriched in theelectrically neutral components and those of opposite charge. Bothstreams can be used as feed streams for further purification orseparation, by balanced flow electrophoresis or other means.Additionally, the balanced flow electrophoresis of the inventionprovides higher yields in terms of absolute concentration than areobtainable with forced flow systems.

The following examples illustrate the invention.

EXAMPLE 1

A balanced flow electrophoresis apparatus is assembled using two cellpairs separated from each other and from the electrodes by semipermeablemembranes. The anode and cathode employed have cross-sectional areas of97 square centimeters. The cells have the same cross-sectional area (97square centimeters) and each chamber in each cell is 0.18 centimetersthick. Filter paper is employed as the boundary membrane. The apparatusis employed to separate albumin from gamma globulin in human bloodplasma. Clear plastic leveling tanks are employed in the outlet lines,and a multiple-channel peristaltic pump is used to provide constant flowpumping in the inlet and outlet lines. During several different runs,the net fluid flow in both streams has been measured by collecting theoutput streams over uniform time intervals such as five or ten minutes.These observations indicated that the fluid flow rates in both streamswere substantially equal (less than about 5 percent difference). In mostcases, no differences would be detected by volumetric measurement ingraduated cylinders.

In one such operation, filtered, undialyzed human plasma, buffered to apH of 6.4 with 0.025 molar phosphate buffer U.S.P. is employed as the Astream. The B stream is aqueous 0.025 molar phosphate buffer, pH about6.35. The cell stack is primed with about 50 milliliters of the samebuffer, and the A and B streams are passed through the cell stack at aflow rate of 1.0 milliliter per minute, the A stream flowing upward andthe B stream flowing downward. The electrodes are connected to a directcurrent power supply to pass a constant current of one ampere at 32-24volts (the voltage decreasing during the separation) through the cellstack. The input temperatures of the A and B streams are about 4° C. atthe reservoirs and the output temperatures between 19° C. and 22° C.

The streams are analyzed for total protein by the biuret reaction.Specific protein contents are assayed by cellulose acetateelectrophoresis. The plasma feed stream A has an initial protein contentof 4.03 grams per liter, of which 64.9 percent, 2.62 gm/l, is albumin.The output stream A is reduced in total protein to 0.90 gm/l so thatabsolute albumin content is 0.45 gm/l (50.0 percent). The output bufferstream B is enriched in protein to an absolute concentration of 2.80grams per liter of which 2 gm/l, 71.4 percent is albumin. (Resultsuncorrected for dilution effect of priming buffer.)

EXAMPLE 2

In another operation the A stream is a mixture of 63.9 percent humanalbumin and 36.1 percent gamma globulin in aqueous 0.025 M phosphatebuffer, pH 6.38, total protein concentration 1.73 gm/liter (1.11gm/liter albumin and 0.62 gm/liter gamma globulin). The B stream isaqueous 0.025 molar phosphate buffer, pH 6.4. Both streams are primedinitially with 50 milliliters of the same buffer before the proteinmixture is introduced. The A stream is fed downward and the B streamupward at 1 milliliter per minute, and the current employed is 1.0ampere at 26 volts. The total output of the A stream is collected,assayed and found to contain 0.34 grams per liter total protein,substantially all gamma globulin. Allowing for dilution with the primingbuffer, this represents a yield of 100 percent gamma globulin in aconcentration of 0.41 grams per liter of starting protein solution, withan increase in the purity of the gamma globulin from 36.1 percent to 100percent.

300 Milliliters of the B stream are found to contain 1.08 gm/liter totalprotein, of which 0.93 gm/liter or 86.3 percent is albumin and 0.15gm/liter or 13.7 percent is gamma globulin. This represents asubstantially complete transfer of the albumin from the A to the Bstream, with an increase in purity of albumin from 63.9 percent to 86.3percent of the protein content.

In a preferred form of operation, both streams, A and B, are fedconcurrently downward through the cell stack, as depicted in FIGS. 1 and3. The following example utilizes apparatus similar to that of Examples1 and 3, with two cell pairs, in a downward concurrent flow separationof blood plasma.

EXAMPLE 3

Blood plasma adjusted to pH 6.35 with a Na₂ HPO₄, NaH₂ PO₄, H₃ PO₄buffer is fed as the A stream at a rate of 0.5 milliliters per minute.This stream contains albumin and alpha, beta and gamma globulins inratios of 59.5, 13.9, 11.4 and 15.2 percent, initially. The B streamemployed is 0.025 Molar phosphate buffer U.S.P., pH 6.35, fed downwardat 0.5 ml/min. The current employed is 1.2 ampere at 37 volts. Theprotein composition of the exit B stream is found to be 84.6 percentalbumin and 6.2, 6.2 and 3.0 percent alpha, beta and gamma globulins.

In a second run at a flow rate of 1.0 ml/min, with 1.00 ampere at 34volts, protein composition of the starting plasma stream is 67.1, 9.4,9.4 and 14.1 percent albumin, and alpha, beta and gamma globulins,respectively. The exit B stream is found to contain no gamma globulin,and 91 percent albumin, the remaining 9 percent being alpha and betaglobulins.

In operations such as those described above, protein precipitation(generally fibrinogen) takes place outside the cell on the holding ofthe output A stream. No significant fibrinogen precipitation or membranefouling is detected in the cell stack 4 itself.

The invention can also be applied to the supernatant liquids from plasmaprotein fractionation methods based upon alcohol precipitation. (See,for example, Cohn et al., U.S. Pat. Nos. 2,390,074 and 2,469,193, and J.Am. Chem. Soc. 68, 469 (1946); Gerlough, U.S. Pat. No. 2,710,293 and2,710,294; and Hink, U.S. Pat. No. 2,958,628.) In such use, thealcoholic supernatant can be fed directly to the unbalanced flowelectrophoresis cell stack 4 after adjustment of pH to between theisoelectric points of the plasma proteins to be separated. Duringelectrophoresis, the alcohol can be displayed out of the A and B streamsinto the cooling solution, if desired, by appropriate choice ofcolloid-impermeable membranes 9 and relative flow rate.

In a particularly useful embodiment, the balanced flow electrophoresisis employed in conjunction with electrodialysis to purify bloodproteins. It has been found that electrodialysis of the albumin enrichedB stream results in selective precipitation of globulins therein, thusimproving purification of the albumin. In general, this process can becarried out by subjecting plasma (as the A stream) to balanced flowelectrophoresis to separate albumin into the buffered B stream, and thenpassing the B stream through a conventional electrodialysis cell havingconventional ion exchange membranes, and separating the resulting liquidfrom the resulting precipitate.

EXAMPLE 4

In a representative operation, undialyzed, buffered human blood plasma,pH of 6.38-6.45, 0.02-0.3 molar phosphate buffer U.S.P. (United StatesPharmacopoeia) is introduced as the A stream into a balanced flowelectrophoresis stack, using similar 0.025 molar buffer as the B stream.Electrophoresis is carried out using concurrent downward flow at a rateof 1 milliliter per minute on both streams, and a direct current fieldof 2.2 amperes at 56 volts. Analysis of an aliquot from the output Bstream indicates a total protein content of 25.2 grams per liter, ofwhich 78 percent is albumin.

The B stream is pumped through an electrodialysis cell comprising ananode compartment wth a ruthenium oxide coated titanium electrode, ananion exchange resin membrane (commercially available from Ionics) acenter compartment for the B stream protein solution, a cation exchangeresin membrane (Ionics) and a cathode compartment with a nickel cathode.Electrode and ion exchange membrane areas are 97 square centimeters.Refrigerated aqueous 0.1 molar solution of sodium sulfate and 0.1 molarsodium thiosulfate is pumped through the electrode compartments as acooling sweep stream. The B stream is circulated through the cell at 400milliliters per minute while a direct current electric field of 5 voltsis applied between the electrodes. The initial current is 0.73 amperes,dropping to 0.02 amperes, by the end of the run. The sweep streamtemperatures if 5°-6° C., and the B stream temperature 7°-9° C.

The output stream is centrifuged and the supernatant liquid is found tocontain 21.4 grams total protein per liter, 86.5 percent albumin.

In a similar operation, a B stream containing 7.9 grams per liter totalprotein, 88 percent albumin, is similarly subjected to electrodialysis.The precipitate which forms during the run is separated bycentrifugation. The supernatant solution is analyzed, and found tocontain 6.5 grams total protein, of which 91.6 percent is albumin.

EXAMPLE 5

In another useful embodiment, the protein solution to be separated issubjected to multiple balanced flow electrophoretic separations.

In apparatus similar to that described in Examples 1-3, filtered,undialyzed human blood plasma was buffered to pH 6.4 with phosphatebuffer. Aliquots were taken for analysis, and 235 milliliters of thebuffered, filtered plasma were used as the A stream. 0.025 Molar, pH 6.4phosphate buffer was used as the B stream. The streams were fed to theelectrophoresis cell at 0.5 ml/minute, concurrently downward, using adirect current of 1.0 ampere. The electrophoresis cell was cooled toprovide temperatures at the inlets and outlets of about 14° C. and about16° C. The output stream B was reserved.

The output of stream A was collected and an aliquot was buffered againto pH 6.4, filtered, and used as the A feed stream with fresh 0.025molar, pH 6.37 phosphate buffer as the B stream, under similarelectrophoresis conditions. Both the A and B streams were collected.

The analyses of the original A feed stream, and the A and B outputstreams from the first and second passes (A, B and A', B', respectively)are set out below in grams per liter.

    ______________________________________                                                 Feed  Outputs                                                                 A     A       A'      B      B'                                      ______________________________________                                        Total protein                                                                            48.0    24.0    11.20 24.8   10.5                                  Gamma-Globulin                                                                           6.82    4.8     3.56  0.99   0.15                                  Beta-Globulin                                                                            4.70    2.14    .82   1.49   0.37                                  Alpha-Globulin                                                                           6.24    2.14    .72   2.48   1.11                                  Albumin    30.24   14.93   6.10  19.84  8.87                                  ______________________________________                                    

In terms of percentage of total protein, the albumin content was 63percent in the original A feed, 80.0 percent in the first B output and84.5 percent in the second B' output, with corresponding reductions ofgamma globulin in the same streams from 14.2, to 4.0 to 1.4 percent.Gamma globulin was purified in the A streams, from 14.2 percent of totalproteins in the feed, to 20.0 percent in the first A output, to 31.8percent in the second, A' output.

The original A feed stream represents about 7.11 grams albumin in thestarting feed stream. On a gross basis (ignoring amounts lost in theapparatus at the ends of runs and amounts not collected during priming)209 ml. of the first B stream and 185 ml. of the second B' stream werecollected. Thus on a gross basis, 5.79 grams, or about 81.5 percent ofthe albumin in the feed, was recovered in the B streams.

What is claimed is:
 1. A method useful for electrophoretic separation ofaqueous solutions containing a mixture of electrically charged proteincomponents, comprising:continuously passing a first solution containingprotein components to be separated and having a pH between theisoelectric points of the proteins to be separated along one surface ofa permeable, non-conducting boundary membrane in an electrophoresiscell; simultaneously passing a second solution along the oppositesurface of said membrane; introducing the first and second solutionsinto the electrophoresis cell on opposite sides of the boundary membraneand continuously withdrawing each solution from the cell after passagealong its corresponding surface of the boundary membrane, the withdrawalof each solution being at a volumetric flow rate approximately equal toits volumetric rate of introduction, and maintaining the hydrostaticpressure of the first solution on one side of the boundary membraneequal to the pressure of the second solution on the opposite sidethereof, whereby net liquid transport across said membrane is minimized;and applying a direct current field across the solutions and themembrane, the polarity of the current being selected to induceelectrophoretic migration of one of the electrically charged proteincomponents of the first solution through the boundary membrane into thesecond solution.
 2. Method of claim 1 wherein the second solution is anaqueous buffer having substantially the same pH as the first solution.3. The method of claim 1 wherein the boundary membrane is oriented in avertical plane, and wherein the linear direction of flow of both liquidsis vertically downward.
 4. The method of claim 1 wherein the firstsolution is blood plasma buffered to a pH between the isoelectric pointsof albumin and gamma-globulin.
 5. The method of claim 4 wherein thesecond solution is an aqueous buffer having a pH substantially the sameas the first solution.